Collagen is a key player in human development, maintenance of health, and a range of common and uncommon diseases. It is considered to be the characteristic structural molecule of the extracellular matrix in multicellular animals. Fibril-forming collagens and basement membrane collagens are ubiquitous in vertebrates and invertebrates, whereas families of more specialized collagens have developed in different organisms.
The collagen structure is defined by the distinctive supercoiled triple-helix conformation, having a (Gly-Xaa-Yaa)n amino acid sequence. In this configuration, Gly provides a glycine residue with Xaa and Yaa independently comprising any known imino or amino acid residue for each repeat unit. Unique properties of the collagen triple-helix motif include its molecular hydrodynamic properties, extensive hydration, ability to bind diverse ligands, and capacity to self-associate to form fibrils and other higher order structures. These distinctive features have been exploited by nature to fill a wide range of structural and functional niches. For example, in humans, characteristic collagen fibrils with an axial D=67 nm period provide the structural backbone of tendons, skin, bone, cartilage, and other connective tissues. A network-like structure of type IV collagen is also important for basement membranes, such as those in the kidney glomerulus and lining of blood vessels.
A high content of hydroxyproline (Hyp) is a unique stabilizing feature found within most animal collagens. Indeed, it is widely believed that Hyp residues stabilize the collagen helical structure so it will not denature when exposed to mammalian body temperatures. Hyp residues are typically formed from the post-translational modification of proline residues at the Yaa positions by the enzyme prolyl hydroxylase. Once modified, Hyp confers a thermal stability that has been shown to be much greater than that conferred by Pro residues, or any other imino or amino acid, alone. Indeed, previously evaluated collagens without any Hyp have been found to be unstable when exposed to mammalian bodily temperatures.
There have been numerous attempts to design biomaterial products utilizing isolated animal-derived collagen. Such products, while functional, give rise to increasing concerns including the risk of contamination by infectious agents, as well as product standardization. Moreover, animal-derived collagen is limited in that extracted collagens cannot be designed to enhance or modify specific biological properties. Accordingly, attention has shifted away from isolation of animal collagen and toward production of recombinant collagens produced within micro-organism models.
Production of recombinant collagen in an industrial quantity has been very difficult because bacterial hosts lack the biological mechanisms for the post-translational modification of proline residues to hydroxyprolines. Notwithstanding, potentially useful triple-helix-containing collagen-like proteins have been identified in a number of bacteria in recent years. In several pathogenic bacteria, collagen-like proteins have been shown to be expressed and to form stable triple-helical proteins which play a role in pathogenicity. For example, Scl1 and Scl2 proteins from bacterium group A Streptococcus pyogenes (GAS) are expressed on the bacterial cell surface, and are thought to mediate GAS internalization by human cell. Even without post-translational modification of proline, Scl1 and Scl2 have been shown to form heat stable triple-helical structures when expressed as recombinant proteins, particularly when expressed with an amino-terminal globular domain (VSp). Other prokaryotic collagen-like have also been characterized and include Bacillus cereus and Bacillus anthracis proteins associated with the exosporium with a probable role in spore-host interactions; pneumococcal collagen-like protein A (PclA) contributing to adhesion and invasion of host cells; and a family of seven collagen-like proteins, called SclC-SclI from Streptococcus equi subspecies, which are expressed upon infection of horses leading to the pathological condition known as strangles.
These bacterial collagen-like proteins offer an opportunity to create stable triple-helix protein products in a high yield bacterial expression system. The bacterial origin of the collagen-like protein ensures compatibility in the bacterial expression system in terms of codon usage and other factors. Beyond the previously identified sequences, a collagen product is desirable that can easily be produced by recombinant methods on a large scale, while providing greater heat stability, the ability refold in vivo after denaturation, and improving the biological use of the final product. Such collagens, could potentially be aggregated and would be used to make various products, to include biomaterials. As provided herein, the present invention addresses the foregoing needs.